Persistent hypoxia promotes myofibroblast differentiation via GPR-81 and differential regulation of LDH isoenzymes in normal and idiopathic pulmonary fibrosis fibroblasts.

Hypoxia, a state of insufficient oxygen availability, promotes cellular lactate production. Lactate levels are increased in lungs from patients with idiopathic pulmonary fibrosis (IPF), a disease characterized by excessive scar formation, and lactate is implicated in the pathobiology of lung fibrosis. However, the mechanisms underlying the effects of hypoxia and lactate on fibroblast phenotype are poorly understood. We exposed normal and IPF lung fibroblasts to persistent hypoxia and found that increased lactate generation by IPF fibroblasts was driven by the FoxM1-dependent increase of lactate dehydrogenase A (LDHA) coupled with decreased LDHB that was not observed in normal lung fibroblasts. Importantly, hypoxia reduced α-smooth muscle actin (α-SMA) expression in normal fibroblasts but had no significant impact on this marker of differentiation in IPF fibroblasts. Treatment of control and IPF fibroblasts with TGF-ß under hypoxic conditions did not significantly change LDHA or LDHB expression. Surprisingly, lactate directly induced the differentiation of normal, but not IPF fibroblasts under hypoxic conditions. Moreover, while expression of GPR-81, a G-protein-coupled receptor that binds extracellular lactate, was increased by hypoxia in both normal and IPF fibroblasts, its inhibition or silencing only suppressed lactate-mediated differentiation in normal fibroblasts. These studies show that hypoxia differentially affects normal and fibrotic fibroblasts, promoting increased lactate generation by IPF fibroblasts through regulation of the LDHA/LDHB ratio and promoting normal lung fibroblast responsiveness to lactate through GPR-81. This supports a novel paradigm in which lactate may serve as a paracrine intercellular signal in oxygen-deficient microenvironments.

The aged extracellular matrix and the profibrotic role of senescence-associated secretory phenotype.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal lung disease that is primarily found in the elderly population, and several studies have demonstrated that aging is the major risk factor for IPF. IPF is characterized by the presence of apoptosis-resistant, senescent fibroblasts that generate an excessively stiff extracellular matrix (ECM). The ECM profoundly affects cellular functions and tissue homeostasis, and an aberrant ECM is closely associated with the development of lung fibrosis. Aging progressively alters ECM components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction through the expression of factors linked to a senescence-associated secretary phenotype (SASP). There is growing evidence that SASP factors affect various cell behaviors and influence ECM turnover in lung tissue through autocrine and/or paracrine signaling mechanisms. Since life expectancy is increasing worldwide, it is important to elucidate how aging affects ECM dynamics and turnover via SASP and thereby promotes lung fibrosis. In this review, we will focus on the molecular properties of SASP and its regulatory mechanisms. Furthermore, the pathophysiological process of ECM remodeling by SASP factors and the influence of an altered ECM from aged lungs on the development of lung fibrosis will be highlighted. Finally, recent attempts to target ECM alteration and senescent cells to modulate fibrosis will be introduced.NEW & NOTEWORTHY Aging is the most prominent nonmodifiable risk factor for various human diseases including Idiopathic pulmonary fibrosis. Aging progressively alters extracellular matrix components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction. In this review, we will discuss the pathological impact of aging and senescence on lung fibrosis via senescence-associated secretary phenotype factors and potential therapeutic approaches to limit the progression of lung fibrosis.

Independent role of caspases and Bik in augmenting influenza A virus replication in airway epithelial cells and mice.

Caspases and poly (ADP-ribose) polymerase 1 (PARP1) have been shown to promote influenza A virus (IAV) replication. However, the relative importance and molecular mechanisms of specific caspases and their downstream substrate PARP1 in regulating viral replication in airway epithelial cells (AECs) remains incompletely elucidated. Here, we targeted caspase 2, 3, 6, and PARP1 using specific inhibitors to compare their role in promoting IAV replication. Inhibition of each of these proteins caused significant decline in viral titer, although PARP1 inhibitor led to the most robust reduction of viral replication. We previously showed that the pro-apoptotic protein Bcl-2 interacting killer (Bik) promotes IAV replication in the AECs by activating caspase 3. In this study, we found that as compared with AECs from wild-type mice, bik-deficiency alone resulted in ~ 3 logs reduction in virus titer in the absence of treatment with the pan-caspase inhibitor (Q-VD-Oph). Inhibiting overall caspase activity using Q-VD-Oph caused additional decline in viral titer by ~ 1 log in bik-/- AECs. Similarly, mice treated with Q-VD-Oph were protected from IAV-induced lung inflammation and lethality. Inhibiting caspase activity diminished nucleo-cytoplasmic transport of viral nucleoprotein (NP) and cleavage of viral hemagglutinin and NP in human AECs. These findings suggest that caspases and PARP1 play major roles to independently promote IAV replication and that additional mechanism(s) independent of caspases and PARP1 may be involved in Bik-mediated IAV replication. Further, peptides or inhibitors that target and block multiple caspases or PARP1 may be effective treatment targets for influenza infection.

A role for Toll-like receptor 3 in lung vascular remodeling associated with SARS-CoV-2 infection.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs). In vitro , SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

Biomechanical Force and Cellular Stiffness in Lung Fibrosis.

Lung fibrosis is characterized by the continuous accumulation of extracellular matrix (ECM) proteins produced by apoptosis-resistant (myo)fibroblasts. Lung epithelial injury promotes the recruitment and activation of fibroblasts, which are necessary for tissue repair and restoration of homeostasis. However, under pathologic conditions, a vicious cycle generated by profibrotic growth factors/cytokines, multicellular interactions, and matrix-associated signaling propagates the wound repair response and promotes lung fibrosis characterized not only by increased quantities of ECM proteins but also by changes in the biomechanical properties of the matrix. Importantly, changes in the biochemical and biomechanical properties of the matrix itself can serve to perpetuate fibroblast activity and propagate fibrosis, even in the absence of the initial stimulus of injury. The development of novel experimental models and methods increasingly facilitates our ability to interrogate fibrotic processes at the cellular and molecular levels. The goal of this review is to discuss the impact of ECM conditions in the development of lung fibrosis and to introduce new approaches to more accurately model the in vivo fibrotic microenvironment. This article highlights the pathologic roles of ECM in terms of mechanical force and the cellular interactions while reviewing in vitro and ex vivo models of lung fibrosis. The improved understanding of the fundamental mechanisms that contribute to lung fibrosis holds promise for identification of new therapeutic targets and improved outcomes.

Pathological effects of nano-sized particles on the respiratory system.

Recent advances in nanotechnology have made significant progress in biomedical sciences. However, there are growing concerns that exposure to naturally-occurring and synthetic nano-sized particles promotes various types of human diseases. The lungs are the vital organ for gas exchange. Due to their unique position, the lungs can be the primary route that nanoparticles (NPs) enter the human body, which exerts toxic effects. In spite of the wide applications of NPs, the precise effects on health following exposure are not fully understood. To address this, the biophysical properties of naturally-occurring and synthetic NPs, their pathological effects on the respiratory system and the potential human health risk are examined. The goal of this review is to provide the current evidence that exposure to NPs is a risk factor to develop human diseases. The understanding of the pathological process by NPs is essential in preventing and the treatment of nanoparticle-induced human diseases.

Fibroblasts from patients with idiopathic pulmonary fibrosis are resistant to cisplatin-induced cell death via enhanced CK2-dependent XRCC1 activity.

Idiopathic pulmonary fibrosis (IPF) is a deadly and progressive fibrotic lung disease, but the precise etiology remains elusive. IPF is characterized by the presence of apoptosis-resistant (myo)fibroblasts that relentlessly produce a collagen-rich extracellular matrix (ECM). Recent studies showed that an anti-cancer chemotherapy drug cisplatin is implicated in the development of pulmonary fibrosis, suggesting that the treatment of cancer patients with cisplatin may alter fibroblast viability. To address this possibility, we investigated the cisplatin-induced cell death mechanism in lung fibroblasts derived from IPF and non-IPF patients in response to a collagen matrix. IPF fibroblasts showed enhanced resistance to cisplatin-induced cell death compared to non-IPF fibroblasts in a time- and dose-dependent manner. Molecular study showed that the expression of γH2AX, PUMA and caspase-3/7 activity was abnormally reduced in IPF fibroblasts, suggesting that DNA damage-induced apoptosis caused by cisplatin was suppressed in IPF fibroblasts. Our study further revealed that DNA repair protein XRCC1 activity was aberrantly increased as a result of CK2 hyper-activation in cisplatin-treated IPF fibroblasts, and this alteration protected IPF fibroblasts from cisplatin-induced cell death. Our results showed that IPF fibroblasts residing in a collagen rich matrix are resistance to cisplatin-induced cell death due to the aberrantly high CK2/XRCC1-dependent DNA repair activity. This finding suggests that pulmonary fibrosis may develop and worsen due to the presence of apoptosis-resistant lung fibroblasts in cisplatin-treated cancer patients.

Hyaluronan-CD44/RHAMM interaction-dependent cell proliferation and survival in lung cancer cells.

Although members of the hyaluronan (HA)-CD44/HA-mediated motility receptor (RHAMM) signaling pathway have been shown to be overexpressed in lung cancer, their role in lung tumorigenesis is unclear. In the present study, we first determined levels of HA and its receptors CD44 and RHAMM in human non-small cell lung cancer (NSCLC) cells and stromal cells as well as mouse lung tumors. Subsequently, we examined the role of HA-CD44/RHAMM signaling pathway in mediating the proliferation and survival of NSCLC cells and the cross-talk between NSCLC cells and normal human lung fibroblasts (NHLFs)/lung cancer-associated fibroblasts (LCAFs). The highest levels of HA and CD44 were observed in NHLFs/LCAFs followed by NSCLC cells, whereas THP-1 monocytes/macrophages showed negligible levels of both HA and CD44. Simultaneous silencing of HA synthase 2 (HAS2) and HAS3 or CD44 and RHAMM suppressed cell proliferation and survival as well as the EGFR/AKT/ERK signaling pathway. Exogenous HA partially rescued the defect in cell proliferation and survival. Moreover, conditioned media (CM) generated by NHLFs/LCAFs enhanced the proliferation of NSCLC cells in a HA-dependent manner as treatment of NHLFs and LCAFs with HAS2 siRNA, 4-methylumbelliferone, an inhibitor of HASs, LY2228820, an inhibitor of p38MAPK, or treatment of A549 cells with CD44 blocking antibody suppressed the effects of the CM. Upon incubation in CM generated by A549 cells or THP-1 macrophages, NHLFs/LCAFs secreted higher concentrations of HA. Overall, our findings indicate that targeting the HA-CD44/RHAMM signaling pathway could be a promising approach for the prevention and therapy of lung cancer.

FoxM1-dependent RAD51 and BRCA2 signaling protects idiopathic pulmonary fibrosis fibroblasts from radiation-induced cell death.

Radiation therapy is critical for the control of many tumors and lung is an important dose-limiting organ that impacts radiation dose prescribed to avoid irreversible pulmonary fibrosis in cancer survivors. Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung disease caused by aberrantly activated lung (myo)fibroblasts. The presence of pro-fibrotic, apoptosis-resistant fibroblasts in IPF promotes progressive fibrosis and may have a role in other diseases, if these resistant cells are selected for as a consequence of treatment. However, the pathological response of IPF fibroblasts to radiation compared to non-IPF lung fibroblasts is not known. To address this, we examined fibroblast viability following radiation in lung fibroblasts from IPF and non-IPF patients and the underlying mechanism that protects IPF fibroblasts from radiation-induced death. IPF fibroblasts are significantly more resistant to apoptosis compared to non-IPF lung fibroblasts, suggesting that resistance to radiation-induced cell death is a predominant mechanism leading to lung fibrosis. Analysis of γH2AX induction demonstrated that radiation-induced DNA damage is reduced in IPF fibroblasts and correlates to the activation of the transcription factor forkhead box M1 (FoxM1) and subsequent upregulation of DNA repair proteins RAD51 and BRCA2. FoxM1 activation occurs secondary to FoxO3a suppression in IPF fibroblasts while restoration of FoxO3a function sensitizes IPF fibroblasts to radiation-induced cell death and downregulates FoxM1, RAD51, and BRCA2. Our findings support that increased FoxO3a/FoxM1-dependent DNA repair may be integral to the preservation of death-resistant fibrotic fibroblasts after radiation and that selective targeting of radioresistant fibroblasts may mitigate fibrosis.

The Role of the Mammalian Target of Rapamycin (mTOR) in Pulmonary Fibrosis.

The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)-dependent pathway is one of the most integral pathways linked to cell metabolism, proliferation, differentiation, and survival. This pathway is dysregulated in a variety of diseases, including neoplasia, immune-mediated diseases, and fibroproliferative diseases such as pulmonary fibrosis. The mTOR kinase is frequently referred to as the master regulator of this pathway. Alterations in mTOR signaling are closely associated with dysregulation of autophagy, inflammation, and cell growth and survival, leading to the development of lung fibrosis. Inhibitors of mTOR have been widely studied in cancer therapy, as they may sensitize cancer cells to radiation therapy. Studies also suggest that mTOR inhibitors are promising modulators of fibroproliferative diseases such as idiopathic pulmonary fibrosis (IPF) and radiation-induced pulmonary fibrosis (RIPF). Therefore, mTOR represents an attractive and unique therapeutic target in pulmonary fibrosis. In this review, we discuss the pathological role of mTOR kinase in pulmonary fibrosis and examine how mTOR inhibitors may mitigate fibrotic progression.

The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

Desensitization of idiopathic pulmonary fibrosis fibroblasts to Alternaria alternata extract-mediated necrotic cell death.

Alternaria alternata is an allergenic fungus and known to cause an upper respiratory tract infection and asthma in humans with compromised immunity. Although A. alternata's effect on airway epithelial cells has previously been examined, the potential role of A. alternata on lung fibroblast viability is not understood. Since lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) display a distinct phenotype that is resistant to stress and cell death inducing conditions, the investigation of the role of Alternaria on pathological IPF fibroblasts provides a better understanding of the fibrotic process induced by an allergenic fungus. Therefore, we examined cell viability of control and IPF fibroblasts (n = 8 each) in response to A. alternata extract. Control fibroblast cell death was increased while IPF fibroblasts were resistant when exposed to 50-100 µg/mL of A. alternata extract. However, there was no significant difference in kinetics or magnitude of Ca2+ responses from control lung and IPF fibroblasts. In contrast, unlike control fibroblasts, intracellular reactive oxygen species (ROS) levels remained low when IPF cells were treated with A. alternata extracts as a function of time. Caspase 3/7 and TUNEL assay revealed that enhanced cell death caused by A. alternata extract was likely due to necrosis, and 7-AAD assay and the use of sodium pyruvate for ATP generation further supported our findings that IPF fibroblasts become resistant to A. alternata extract-induced necrotic cell death. Our results suggest that exposure to A. alternata potentially worsens the fibrotic process by promoting normal lung fibroblast cell death in patients with IPF.

Advanced Therapeutic Strategies for Chronic Lung Disease Using Nanoparticle-Based Drug Delivery.

Chronic lung diseases include a variety of obstinate and fatal diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), and lung cancers. Pharmacotherapy is important for the treatment of chronic lung diseases, and current progress in nanoparticles offers great potential as an advanced strategy for drug delivery. Based on their biophysical properties, nanoparticles have shown improved pharmacokinetics of therapeutics and controlled drug delivery, gaining great attention. Herein, we will review the nanoparticle-based drug delivery system for the treatment of chronic lung diseases. Various types of nanoparticles will be introduced, and recent innovative efforts to utilize the nanoparticles as novel drug carriers for the effective treatment of chronic lung diseases will also be discussed.

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3.

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3ß function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL-induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL-mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK-3ß-NFATc1 and protects IPF cells from the FasL-dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14.

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.

Alteration of Aging-Dependent MicroRNAs in Idiopathic Pulmonary Fibrosis.

Preclinical Research Idiopathic Pulmonary Fibrosis (IPF) is the most severe fibrotic lung disease and characterized by the accumulation of (myo)fibroblasts and collagen within the alveolar wall resulting in obliteration of the gas-exchange surface. Although the detailed pathogenesis is not understood, recent studies have found that several microRNAs (miRNAs) are associated with the progression of lung diseases including IPF. IPF is a fibrotic disease and, most frequently found in an aged population. In this review, the functional roles of miRNAs that are deregulated in IPF progression are discussed together with how aging affects the miRNA signature, altering the fibroblast phenotype and promoting lung fibrosis. Finally, the possibility of targeting miRNAs as a therapeutic approach for the treatment of IPF is discussed.

Reduced FoxO3a expression causes low autophagy in idiopathic pulmonary fibrosis fibroblasts on collagen matrices.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease, and fibroblasts derived from patients with IPF are resistant to type I collagen matrix-induced cell death. The alteration of the PTEN-Akt axis permits IPF fibroblasts to maintain a pathological phenotype on collagen by suppressing autophagy. However, the precise underlying mechanism by which the Akt downstream molecule suppresses autophagic activity remains elusive. FoxO3a is a direct target of Akt and is implicated with the transcriptional activation of autophagy. Therefore, we investigated whether reduced FoxO3a expression causes abnormally low autophagy in IPF fibroblasts on collagen. We found that FoxO3a mRNA and protein levels are low in IPF fibroblasts, which subsequently suppresses the autophagosomal marker LC3B expression on collagen matrix. In contrast, the majority of control fibroblasts showed an increase in FoxO3a and LC3B expression at both the mRNA and protein levels. The luciferase assay confirmed that FoxO3a binds to the promoter region of LC3B and transcriptionally activates LC3B. The overexpression of wild-type FoxO3a increased LC3B mRNA and protein expression in IPF fibroblasts, whereas the dominant negative FoxO3a decreased the LC3B level in control fibroblasts. The inhibition of autophagic activity sensitized control fibroblasts to collagen matrix-induced cell death. In contrast, enhanced viability was found when autophagic function was inhibited in IPF fibroblasts. Our study showed that aberrantly low FoxO3a expression participates in reducing autophagic activity via transcriptional suppression of LC3B in IPF fibroblasts on collagen. This suggests that low autophagic activity by the alteration of FoxO3a may contribute to IPF progression.

FoxO3a and disease progression.

The Forkhead box O (FoxO) family has recently been highlighted as an important transcriptional regulator of crucial proteins associated with the many diverse functions of cells. So far, FoxO1, FoxO3a, FoxO4 and FoxO6 proteins have been identified in humans. Although each FoxO family member has its own role, unlike the other FoxO families, FoxO3a has been extensively studied because of its rather unique and pivotal regulation of cell proliferation, apoptosis, metabolism, stress management and longevity. FoxO3a alteration is closely linked to the progression of several types of cancers, fibrosis and other types of diseases. In this review, we will examine the function of FoxO3a in disease progression and also explore FoxO3a's regulatory mechanisms. We will also discuss FoxO3a as a potential target for the treatment of several types of disease.